Novel norovirus recombinants detected in South Africa

From 2010 to 2012, NoV GII-positive stool specimens were selected for further genotypic characterisation. All specimens were received from the Rotavirus Sentinel Surveillance Programme which routinely screens stool specimens for enteric pathogens. Specimens were received from children up to the age of 5 years who were hospitalised with severe gastroenteritis in four provinces of SA: Gauteng (Johannesburg), KwaZulu-Natal (Empangeni and Pietermaritzburg), Mpumalanga (Bushbuckridge) and the Western Cape (Cape Town). In 2013, a NoV GII-positive stool specimen from a sporadic case of gastroenteritis in an adult was also characterised. Stool suspensions (10%) were prepared in ultrapure water (Adcock Ingram, Johannesburg, SA) and stored at −20°C until nucleic acid extraction.

For amplification of partial RNA polymerase (region A) and capsid (region C) gene regions, nucleic acid was extracted from 200 μl stool suspension using the MagNA Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics GmbH, Mannheim, Germany) on the automated MagNA Pure system (Roche Diagnostics). For amplification of a region spanning partial polymerase and capsid genes, nucleic acid was extracted from 140 μl stool suspension using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany). Nucleic acid was eluted in 50–60 μl and stored at −70°C until use.

Reverse transcription was performed using 10 μl extracted RNA and 50 U RevertAid™ Premium reverse transcriptase (Thermo Scientific, Waltham, MA), with 30 μM random hexamer primers. Region A (polymerase gene) was amplified using 5 μl of cDNA in a 50 μl reaction containing 200 μM dNTPs, 0.3 μM primers JV12Y and JV13I [34], 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA) and 1.5 mM MgCl₂. The reaction conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 30 sec, 37°C for 1 min 30 sec, 72°C for 1 min and a final step at 72°C for 5 min. Region C (capsid gene) was amplified using published primers G2SKF and G2SKR [35] as previously described [22]. The overlap region (1090 bp), spanning sections of the polymerase and capsid genes including the suspected recombination breakpoint, was amplified using primers JV12Y and G2SKR. Briefly, the 50 μl reaction contained 0.5 μl cDNA, 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems), 200 μM dNTPs, 0.3 μM primer JV12Y and 1 μM primer G2SKR, using the following cycling parameters: 95°C for 10 min, 40 cycles of 94°C for 30 sec, 37°C for 1 min and 72°C for 2 min, followed by 72°C for 5 min. For strains that could not be genotyped based on partial polymerase or capsid sequences (remained unassigned phylogenetically), complete polymerase or capsid genes were amplified, respectively. For amplification of the complete polymerase gene, the following primer was designed: GIIpolF 5′-GTC ATC TGT GCA ACA CAA GG-3′ and used in conjunction with JV13I to amplify a 1038 bp region of ORF1. The 50 μl reaction contained 1.25 U AmpliTaq Gold DNA polymerase, 200 μM dNTPs, 0.5 μM of GIIpolF and 0.3 μM JV13I. The following cycling parameters were used: 95°C for 10 min, followed by 40 cycles of 95°C for 30 sec, 37°C for 1 min 30 sec and 72°C for 2 min, and a final extension at 72°C for 5 min. A consensus sequence was created using this amplicon and the overlap amplicon to obtain the complete RNA polymerase gene (1533 bp). A two-step semi-nested RT-PCR was used to amplify the 1623 bp capsid gene. In the first reaction, 1 μl of cDNA was combined with 1.25 U AmpliTaq Gold DNA polymerase, 200 μM dNTPs, primers QNIF2 [36] (200 nM) and GII.4 capsid reverse (5′-CCA TTA TAA WRC WCG YCT RCG CC-3′) (600 nM) and PCR buffer containing 1.5 mM MgCl₂. One microliter of the first round PCR product was used as template in the second PCR, which used the same reaction mix except for forward primer G2SKF. The following cycling parameters were used: 95°C for 10 min, 35 cycles of 94°C for 45 sec, 58°C for 1 min and 72°C for 1 min 40 sec, and a final extension at 72°C for 10 min. All primers were manufactured by Applied Biosystems.

Amplicons from the partial RNA polymerase and capsid gene regions were sequenced directly with the ABI PRISM BigDye® Terminator v.3.1 Cycle Sequencing kit (Applied Biosystems) on an ABI 3130 automated analyser (Applied Biosystems). The overlap amplicon, partial ORF1 and complete capsid amplicons were cloned before sequencing, using the CloneJET™ PCR cloning kit (Thermo Scientific). At least two randomly selected clones were sequenced using pJET1.2/blunt specific primers (Thermo Scientific).

Nucleotide sequences were edited and analysed using Sequencher™ 4.9 (Gene Codes Corporation, Ann Arbor, MI), BioEdit Sequence Alignment Editor (V.7.0.9.0) [37] and BLAST-n [38]. The polymerase and capsid genotypes were determined using the Norovirus Genotyping Tool [2] as well as phylogenetic analysis performed in MEGA6 [39]. Sequences were aligned with reference strains using MAFFT version 6 (http://​mafft.​cbrc.​jp/​alignment/​server/​index.​html) and phylogenetic analysis was performed using the neighbor-joining method, validated by 1000 bootstrap replicates. Genotypes were assigned based on clustering with reference strains in the phylogenetic tree with >70% bootstrap support.

Selected NoV strains that clustered with different genotypes based on phylogenetic analysis of the polymerase and capsid sequences were subjected to further recombination analysis. Putative recombination breakpoint analysis was performed using SimPlot version 3.5.1 and the maximum x² test as implemented in RDP version 4.33 [40].

Sequences were submitted to GenBank under the following accession numbers: KC962457-62; KJ710245-48; KM025143.